The transplantation of induced pluripotent stem cells into the cochleae of mature mice

Abstract

Objective: Stem cell transplantation is an effective method for treating sensorineural hearing loss (SNHL), but its safety needs further study. This study aimed to reveal the differentiation outcome of induced pluripotent stem cells (iPSCs) after they were transplanted into cochleae.

Methods: iPSCs were labelled with CM-Dil and identified by flow cytometry. Twenty 6-8-week-old ICR mice were divided into experimental (A) and control (B) groups. Ten mice were microinjected with CM-Dil-labelled iPSC suspension (group A) or an equal volume DMEM (group B) into the left ear cochlea. The tthresholds of all mice were tested by auditory brainstem response (ABR) at 1 week pre-surgery and 4 weeks post-surgery. Differentiated cells were identified by immunohistochemical staining for neuronal cell markers (nestin, neurofilament-M), and teratoma formation was determined by HE staining.

Results: The ABR thresholds in groups A and B at one week pre-surgery (24.50±5.50 vs. 26.00±6.15 dB SPL) and at 4 weeks post-surgery (70.50±4.97 vs. 68.00±5.37 dB SPL) were not significantly different; however, in both groups, the thresholds were lower at pre-surgery than at 4 weeks post-surgery. In group A, CM-Dil-labelled iPSCs were observed in the cochlear perilymph, endolymph, and modiolus, and some red fluorescence-labelled cells expressed neural cell markers. In group B, no fluorescence was observed in the cochleae, but teratomas were observed in some cochleae. A teratoma was observed in each of two cochleae after iPSCs transplantation by HE staining.

Conclusion: Mouse iPSCs can differentiate into cells with neuronal cell markers 4 weeks post-cochlear transplantation, and transplanted undifferentiated iPSCs may form teratomas. However, in the short-term, hearing loss in mice caused by cell transplantation through round window pathways cannot be improved by cochlear iPSC transplantation.

Keywords: Induced pluripotent stem cells (iPSCs); cell differentiation; inner ear transplantation; spiral ganglion neuron; teratoma.

Figure 1

A. Mouse induced pluripotent cells (miPSCs) (Scale bar = 500 um). B. miPSCs that form cell groups with a strong edge refraction were undifferentiated (Scale bar = 50 um). C. Cells display a strong red fluorescence after CM-Dil labelling (Scale bar = 500 um). D. Labelling efficiency of CM-Di1 indicated by results of flow cytometry.

Figure 2

ABR hearing threshold was (A) 20 dB SPL at one week pre-surgery and (B) 70 dB SPL before the mice were sacrificed at four weeks post-surgery.

Figure 3

In vivo iPSC differentiation. CM-Di1-positive iPSCs were found in the scala tympani, scala media, modiolus, and scala vestibule (arrowheads in E). Blue fluorescence shows nuclear labelling with 4’,6-diamino-2-phenyl-indole (DAPI). Inside the modiolus, red fluorescence-labelled iPS cells express neuronal markers, neurofilament-M. Scale bars = 100 μm (A-D), 500 μm (E). ST, scala tympani; SM, scala media; SV, scala vestibuli; MO, modiolus.

Figure 4

In vivo iPSC differentiation. Immunohistochemical analysis of neuronal markers (nestin) in ICR mouse cochleae at 4 weeks after transplantation. CM-Di1-positive iPSCs were found in the modiolus (arrowheads in E). Blue fluorescence shows nuclear labelling with 4’,6-diamino-2-phenyl-indole (DAPI). Inside the modiolus, red fluorescence-labelled iPSCs expressed neuronal markers, nestin. Scale bars = 100 μm (A-D), 500 μm (E). ST, scala tympani; SM, scala media; SV, scala vestibuli; MO, modiolus.

Figure 5

Teratoma formation at 4 weeks post transplantation. The specimen was fixed with 4% paraformaldehyde (PFA), embedded with OCT compound, sectioned, and stained with hematoxylin and eosin (HE). (A, D) A midmodiolar section of the cochlea. (B, C) Higher magnification of (A). (E) Higher magnification of (D). Arrows in B, C, and E indicate undifferentiated cells, mesenchymal cells, and ducts, respectively. Scale bars = 25 μm (A), 50 μm (D), 100 μm (B, C, E). ST, scala tympani; SM, scala media; SV, scala vestibuli; MO, modiolus.